Is it possible to perform a PCR, using as the reverse primer a oligo dT, the same used in the reverse trancription? I am trying to amplify a mRNA, but with no success
You can use any primer including oligodT as long as your template has binding site for it or the mismatch between the primer and the template is at the 5'end of the primer.
When using mRNA, you first need to make cDNA out of it. Also keep in mind that the quality of your cDNA would be critical factor in PCR. When you use oligodT as reverse primer , you are starting at 3'end. Choosing a forward prime at very 5 ' end of your sequence would need that cDNA is of very high quality (i.e. You have full length transcripts). If you are not sure, then choose a forward primer as close as possible to the 3' end. Moreover, not to forget are the slippage problems almost every polymerase has with homopolymeric sequences.
I hope this information helps.
Yes it is possible
- it is the basis of a technique called rapid amplification of cDNA ends or RACE
- However this technique is technically more challenging than standard PCR so unless you are trying to characterise the 3’ ends if your transcript my advice would be to design another reverse primer just upstream of your poly A tail and then perform standard PCR
- even RACE is done by initially amplifying your polyA and with oligo dT but with a linker adaptor attached so you then perform secondary PCR with a reverse primer to that adaptor
- far easier to make a reverse primer at the very end of your transcript or make a series of primers to alternative transcript 3’ ends
- RACE is normally used to fish out different transcript isoforms of your gene in that tissue but unless you really need to know this you should simply design a primer to the last exon in all known transcripts predicted by Ensembl and carry out standard PCR
Thank you so much. The problem is that I am trying to clone the full length mRNA, with no success. I have tried several protocols, but maybe the transcript is not in great amounts inside the cells.
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